Process for the production of polysaccharide beads

ABSTRACT

The present invention relates to a method for producing porous polysaccharide beads, comprising: (a) mixing a water-based solution of a polysaccharide under stirring with an essentially water-immiscible first organic phase to form an emulsion which separates into two continuous phases; (b) adding a second organic phase comprising an emulsifier and an organic solvent to form a three-phase system; (c) allowing the three-phase system to emulsify; and (d) decreasing the temperature below the gelling point of the polysaccharide, thereby obtaining beads having two sets of pores, said method being characterised in that the emulsifier is a water-insoluble polymer capable of stabilizing said three-phase system.

This application is a 371 of PCT/SE99/01663, filed Sep. 23, 1999.

TECHNICAL FIELD

The present invention relates to methods for producing porouspolysaccharide beads.

BACKGROUND ART

Polysaccharide gels are known to play an important role in themanufacture of materials for separation of mixtures of biomolecules.Among the characteristics making these gels interesting can be mentionedtheir inertness in contact with proteins and other biomolecules, andtheir porous structure. A further important property is their resistanceagainst alkaline conditions, which is of great importance in large scaleseparation processes requiring frequent regeneration and sterilisationof the gel. Polysaccharide gels, unlike many other separation materials,allow in situ regeneration with alkaline agents, e.g. sodium hydroxide.

In many chromatography methods, such as gel filtration, ion exchangechromatography and affinity chromatography, polysaccharide gels arepreferred because of their inertness and well established derivatisationchemistry. However, polysaccharide gels also exhibit certain drawbacks,like the limited mechanical stability. This is not a real problem whencomparatively large gel beads. (around 100 μm) are used in traditionalchromatography. The situation is different when attempts are made toincrease the separation efficacy by the use of smaller gel beads (5-20μm). At flow rates optimal for diffusion, the pressure drop in the gelbed is so high that the polysaccharide beads will collapse.

As a solution to the problem how to increase the separation efficacy,polysaccharide gels which contain two types of pores, small diameterdiffusion pores (micropores), and large diameter flow-trough pores(macropores or superpores), have been disclosed (WO 93/19115). Such“superporous” gels are manufactured by a method wherein a water-basedsolution of the polysaccharide is mixed, under controlled stirring, withan essentially water-immiscible organic phase to form an oil-in-wateremulsion, which when allowed to solidify forms a network of twocontinuous phases; an aqueous polysaccharide phase and an organic phase.The aqueous phase forms a solid matrix, while the organic phase forms anetwork of flow-through pores within the matrix. Simultaneously,conventional small diameter diffusion pores are formed in the aqueousphase.

In order to obtain polysaccharide beads, the oil-in-water emulsion isemulsified in a second organic phase to give a three-phase system,comprising polysaccharide droplets in the organic phase. When thetemperature is decreased below the gelling temperature of thepolysaccharide, beads having the said two types of pores are obtained.In addition to smaller pores (diffusion pores normally around 20 to 500Å), which are typical for polysaccharides, the formed gel thus alsocontains larger pores (“superpores” or “flow-through pores, normallybetween 5 and 100 μm).

When this superporous material is packed in a chromatographic column,some of the flow will pass via the superpores. The biomolecules to beseparated are thus transported also to the inner parts of the beads byconvective flow, which is a faster way of transportation than diffusion.Only short distances will have to be covered by diffusion in thesuperporous material and the particles will therefore, in spite of amuch larger particle size, be as effective as conventionalpolysaccharide beads, and still give rise to a much lower pressure dropover the gel bed.

However, the presently known method for producing superporous gels hasthe drawback that this kind of three-phase system is unstable and has tobe immediately stabilised by cooling in order to form superporous beads.There is thus a need for an improved method for manufacture ofsuperporous gels or beads, which provides increased stability during theemulsifying process and which allows for large-scale production.

DISCLOSURE OF THE INVENTION

It has surprisingly been found that superporous polysaccharide beads asdefined above can be conveniently produced by a method in which theabove-mentioned drawbacks can be minimized.

Consequently, in a first aspect this invention provides a method forproducing porous polysaccharide beads, comprising

(a) mixing a water-based solution (aqueous solution) of a polysaccharideunder controlled stirring with an essentially water-immiscible firstorganic phase to form an emulsion (emulsion 1) which when allowed tosolidify separates into a network of two continuous phases;

(b) adding a second organic phase comprising an emulsifier I and anorganic solvent to form a three-phase system;

(c) allowing the said three-phase system to emulsify (formation ofemulsion 2); and

(d) decreasing the temperature below the gelling point of the saidpolysaccharide, thereby obtaining beads having two sets of pores asdefined above.

The method is characterised in that emulsifier I is a water-insolublepolymer capable of stabilising emulsion 2.

The two-phase system obtained in step (a) (Emulsion 1) can be producedby methods known from WO 93/19115. Briefly, a water-based polysaccharidephase is mixed, under vigorous stirring, with an essentiallywater-immiscible organic phase, consisting of one or more components,usually an organic solvent preferably together with an emulsifier of thetype stabilising “oil-in-water” systems (emulsifier II).

The skilled person will be able to determine by known methods whetherEmulsion 1 will be suitable for preparation of superporous beads. Rapidtests are available for verifying that a given combination of featureswill give a system suitable for the preparation of the desired material.One test involves study of Emulsion 1 under a microscope. The crucialfactor is that the two phases must form a network of two continuousphases upon cooling. Another test involves a function test of pore flow.A small sample of Emulsion 1 is solidified and a thin (about 1 mm) gelslice is prepared. The gel slice is placed on a supporting net and awater jet is directed to the gel surface. If suitable super pores arepresent, the water jet will readily displace the organic phase in thegel slice. The appearance of the gel slice will change from white tosemi-transparent in this process.

Emulsifier I stabilises emulsion 2 by being capable of adsorbing to thedroplets of emulsion 1 in the second organic phase, thereby preventingdestabilisation of the droplets by diffusion of the inner oil phase tothe outer oil phase.

There is a large number of water-insoluble polymers that can be used asemulsifier I. Each candidate to be used can be prechecked, for instanceby running the experimental protocol outlined in the experimental part.The proper functioning of a candidate will give rise to beads withflow-through pores also after prolonged emulsification. Flow-throughpores can be detected either by ESEM (Environmental Scanning ElectronMicroscopy) or simply by visual inspection of the beads. Visualinspection: Transparent beads means that flow-through pores are absentwhile “milky” beads means that flow-through pores are present.

Initially the following water-insoluble polymers were of potentialinterest: Polybutadiene, polyisoprene, polyisobutene, polyacrylates,polymetacrylates, poly(vinyl acetate), polystyrene, polypropene oxide,polytetrahydrofurane, polycarbonate, non-crystalline polyesters,polydimethylsiloxan, ethyl cellulose, cellulose ethers,PEG-polyhydroxystearic acid block copolymers (Hypermer),styrene-butadiene/isoprene block copolymers (Kraton), water-insolubleethylene oxide-propylene oxide block copolymers (Pluronics), ethyleneoxide-tetrahydrofuran block copolymers (Berol).

Preferably, emulsifier I is selected from ethyl celluloses or polyvinylacetates or other polymers having similar balance between hydrophilicityand hydrophobicity.

Polymers used as emulsifier I, should preferably contain at least 20,such as at least 30, monomeric units. When the polymer is an ethylcellulose or a polyvinylacetate, the molecular weight of the polymershould preferably be above 7000 Da.

The organic solvent used in the first and second organic phase (step aand b, respectively) can e.g. be cyclohexane, toluene or heptane, or amixture. Preferably, the said organic solvent is toluene. The organicsolvent used in step a and step b may be the same or different.

It is an important aspect of the invention that the said three-phasesystem can be allowed to emulsify during a time period of more than 40min, preferably around 1 hour, before the temperature is decreased belowthe gelling point of the said polysaccharide.

Different polysaccharides can be used in the methods according to theinvention for production of superporous beads. Examples of suitablepolysaccharides are agar, agarose, alginate, dextran, carrageenan,chitosan, cellulose and starch, as well as mixtures of these. The actualchoice of polysaccharide will depend on the desired properties of thefinal product, for instance with regard to pore size, charge, stabilityin various media, cost, etc. Agarose is preferred, since it isessentially non-ionic and inert towards proteins.

If so required the polysaccharide is derivatized to have the propersolidifying/gelling properties.

As mentioned above, the obtained superporous beads have two sets ofpores. The term “two sets of pores” is intended to mean that the formedpolysaccharide beads comprise

(1) a first set of pores (superpores) essentially being above the sizeof diffusion pores, for instance between 5 and 100 μm, and

(2) a second set of pores essentially being diffusion pores, forinstance with a size between 20 and 521 Å.

Typically the ratio between the pore diameters of the first set of poresand the particle diameter is in the interval 0.01-0.3, with preferencefor 0.05-0.2. The ratio between the pore diameters of the second set andthe particle diameter may in the preferred variants extend up to 0.05but is otherwise below 0.01.

The polysaccharide beads manufactured according to the invention havethe same utility as the known gels disclosed in WO 93/19115. They can beproduced in various shapes, e.g. more or less regular beads, and can beused in the manufacture of chromatographic media and as a carrier matrixfor various biomolecules, like cells, enzymes, antibodies, etc. Becauseof its improved properties, superporous beads are particularly usefulfor high-performance liquid chromatography (HPLC).

After cooling the formed gel may be cross-linked and/or furtherderivatized in the same manner as described in WO 93/19115. Furtherderivatization encompasses among others that affinity groups areattached to the beads. An affinity group is a member of an affinitypair. Well-known affinity pairs are

(a) positively and negatively entities (ion exchange; the immobilisedentity being selected among primary, secondary, tertiary and quaternaryammonium, sulphonate, sulphate, phosphonate, phosphate, carboxy etcgroups),

(b) antibodies and antigens/haptens,

(c) lectins and carbohydrate structures,

(d) IgG binding proteins and IgG,

(e) pair of hydrophobic groups,

(f) polymeric chelators and chelates,

(g) complementary nucleic acids, etc.

Affinity members also include entities participating in catalyticreactions, for instance enzymes, enzyme substrates, cofactors,cosubstrates etc: Members of affinity pairs include chemically producedmimetics of biomolecules.

Potentially affinity groups may also be attached to the polysaccharidebefore it is used for preparing beads according to the invention.

EXAMPLES Synthesis of “Emulsion 1”

Solution A:

An agarose solution is prepared in a batch reactor by adding 40 gagarose to 600 ml distilled water under stirring at +95° C. After 2hours, the solution is cooled to +70° C. NaOH (3 ml, 50%) and 8 mlallylglycidyl ether are added to the agarose solution. The reaction isallowed to continue for 2 hours under stirring at +70° C. The solutionis then neutralised with acetic acid (pH 7-8).

Solution B:

10 g of the surfactant polyoxyethylene sorbitan mono-oleate (Tween 20™)is suspended in 150 ml cyclohexane under stirring at +70° C. Immediatelybefore further use it is stirred vigorously.

Solution B is then mixed with 320 g of Solution A in the batch reactorunder stirring (700 rpm) for 10 minutes at +70° C. A white viscousemulsion (“Emulsion 1”) is obtained.

Synthesis of Superporous Beads

The second organic phase (the emulsion media) is made in an emulsionsreactor by adding 18 g ethyl cellulose (N-50 emulsifier) to 450 mltoluene under stirring at +70° C. The dissolving of N-50 in toluenetakes approximately 2 hours.

The stirring is adjusted to 90 rpm. “Emulsion 1” is transferred to theemulsion media, whereby drops of “Emulsion 1” are formed. After 1 hourof emulsification, the mixture is cooled during approximately 30 min tobelow +25° C. Spherical, superporous particles are obtained.

The gel particles are washed under stirring with ethanol 99.5% witch isdecanted. The gel is then washed on a glass filter with ethanol 99.5%and distilled water. The particles were analysed by ESEM (EnvironmentalScanning Electron Microscopy) which showed that the gel particlescontained flow-trough pores (superpores). Visual inspection showed thatthe beads were milky and non-transparent.

It is clear from the above example that emulsification and cooling ofthe three-phase system could be allowed to take place during arelatively long time (1 hour and 30 min, respectively). This is incontrast to the known method for preparing superporous gels, disclosedin WO 93/19115. In Example 3 of WO 93/19115, 100 ml of “Emulsion 1” ispoured into 200 ml cyclohexane containing 4% (v/v) Span 85 (sorbitantrioleate) as the detergent. With this method, the mixture had to becooled immediately (after only 0.5 min of emulsification) in order forsuperporous beads to be formed.

Consequently, the method according to the present invention is superiorcompared to the previously known method as it provides increasedstability during the emulsifying process, which makes it more suitablefor large-scale synthesis of superporous beads.

Experiments Made During the Priority Year

The same experimenatl protocol as for ethyl cellulose above was carriedout for Poly(vinyl acetate) having Mw 83 000 and 50 000 (AldrichChemical Company, Inc., U.S.A.).

Solution A:

300 ml distilled water, 20 g agarose, 1,5 ml 50% NaOH (w/w), 4 ml allylglycidyl ether.

Solution B:

12 g Tween 20, 150 ml cyclohexane.

Second organic phase:

105 g poly(vinyl acetate) (Mw 83 000) or 40 g poly(vinyl acetate) (Mw 50000) in 225 ml toluene plus 100 ml ethylene dichloride.

The resultant beads were “milky” and nontransparent indicating presenceof flow-through pores.

Experiments were also run with poly(butadiene) (Mw 4500) (JanssensChimica, Belgium) and Polystyrene (Mw 20 000) (Polyscience Inc., USA).In both cases the beads obtained were transparent indicating thatessentially no flow-through pores had been formed.

What is claimed is:
 1. A method for producing porous polysaccharidebeads, comprising (a) mixing a water-based solution of a polysaccharideunder stirring with an essentially water-immiscible first organic phaseto form an emulsion which separates into two continuous phases; (b)adding a second organic phase comprising an emulsifier and an organicsolvent to form a three-phase system; (c) allowing the three-phasesystem to emulsify; and (d) decreasing the temperature below the gellingpoint of the said polysaccharide, thereby obtaining beads having twosets of pores, wherein the emulsifier in said method is awater-insoluble polymer capable of stabilizing said three-phase systemfor a time period of more than 40 minutes, wherein said emulsifier isselected from ethyl cellulose or poly(vinyl acetate).
 2. The methodaccording to claim 1, wherein the formed polysaccharide beads compriseone set of pores with a size between 20 and 500 Å, and another set ofpores with a size between 5 and 100 μm.
 3. The method according to claim1, wherein the three-phase system is emulsified for a time period ofmore than 40 minutes.
 4. The method according to claim 1, wherein thepolysaccharide is agarose.
 5. The method according to claim 1, whereinthe organic solvent is toluene.